All animal use procedures were based on the policies from the Canadian Council on Animal Care and approved by the University of Manitoba Fort Garry Campus Animal Care Committee (for ectothermic fish and rat) or the Stanford Administrative Panel on Laboratory Animal Care Committee in accordance to all policies of Stanford University (tuna).

Tissues were rinsed in ice-cold isolation medium and diced to facilitate homogenization. Two isolation media were used in this study. For tuna, we used 140 mM KCl, 20 mM HEPES, 2 mM EGTA, pH 7.0 at 20°C whereas 120 mM KCl, 20 mM HEPES, 1 mM EGTA, pH 7.1 at 20°C was used for the rats and carp. For rainbow trout and sturgeon we tested both isolation media and found no difference in results. All steps were maintained at 0–4°C. The procedure for rats (Affourtit et al., 2012) and the ectothermic fishes (Banh et al., 2016) are described in detail elsewhere but, briefly, involved incubating diced tissue with a protease followed by disruption by homogenization and isolation of mitochondria by differential centrifugation. The final pellet was resuspended in a small volume of isolation medium at ~20–45 mg mitochondrial protein · ml⁻¹ (determined by biuret assay with BSA as standard).

For tuna, diced tissue was mixed with ice-cold homogenization medium (isolation medium plus 0.5% w/v BSA) and homogenized with a motor-driven Teflon (Polytetrafluoroethylene) to glass homogenizer. The homogenate was centrifuged for 5 min at 900 × g. The supernatant was collected and filtered through a fine plastic mesh to aid in lipid and particulate removal. Following filtration, the supernatant was centrifuged at 9,000 × g for 10 min. The resulting pellet was washed by resuspending in isolation medium followed by centrifugation 9,000 × g for 10 min. Care was taken to not transfer any of the lipid in the supernatant or resuspend any material that was clearly not mitochondria (for example red blood cells and particulate matter). The resulting pellet was washed a second time (as described above) and the final pellet was resuspended in a small volume of isolation medium at ~30–60 mg mitochondrial protein · ml⁻¹ (determined by biuret assay with BSA as standard).

Combined superoxide and hydrogen peroxide production by mitochondria was measured spectrofluorometrically as H₂O₂ efflux in the same medium as used for respiration. In some cases phosphate was omitted or 1 μg · ml⁻¹ oligomycin was added, we found these had relatively little (c. 10% or less) influence on the observed rates and thus pooled all data. The assay medium also contained an H₂O₂ detection system consisting of 5 IU · ml⁻¹ of horseradish peroxidase, 25 IU · ml⁻¹ superoxide dismutase, and 50 μM Amplex UltraRed in a total volume of 2 ml using either an Agilent Eclipse or a Shimadzu RF-5301PC. Fluorescence was monitored, with constant stirring, at 560 nm excitation and 590 nm emission wavelengths, respectively. Raw fluorescence values were transformed to moles of H₂O₂ based on calibration with known amounts of H₂O₂. Background rates of product formation prior to the addition of substrate were low (typically < 10% of rate with substrate) and were subtracted from rates in the presence of substrate.

Mitochondrial substrate oxidation was measured in a water-jacketed chamber with a Rank Brothers Clark-type oxygen electrode (Cambridge, U.K) or using the Oroboros O2K (Innsbruck, Austria). Tuna mitochondria used the following respiration medium: 140 mM KCl, 20 mM Hepes, 1 mM EGTA, 5 mM K₂HPO₄, and 0.5% (w/v) BSA, pH 7.3 at 20°C. For ectotherm fish and rats the standard medium was 120 mM KCl, 20 mM Hepes, 1 mM EGTA, 5 mM K₂HPO₄, and 0.3% (w/v) BSA, pH 7.4 at 20°C. Note, we found comparable (within 10%) results between the two respiration media with the ectothermic fish mitochondria.

Following addition of mitochondria to the chamber the rates of oxygen consumption were measured under the following respiratory conditions: (i) non-phosphorylating, in the presence of exogenous substrate (state 2); (ii) phosphorylating, the rate with exogenous substrate plus 500 or 800 μM ADP (state 3); (iii) a pharmacologically induced non-phosphorylating state, with 1 μg · ml⁻¹ oligomycin (which blocks ATP synthesis) (state 4o). These conditions were measured in sequence and the respiratory control ratios (RCR) were calculated as the rate of oxygen consumption of mitochondria in state 3/state 4o.

All data are shown as mean ± SEM. Values were compared either by ANOVA (with Student-Newman–Keuls post-hoc test) or t-test (Welch's) with p < 0.05 being considered significant.

To determine the FEL we calculated values based on:

Where H₂O₂ efflux rate is in nmol min⁻¹ mg protein⁻¹ and O₂ consumption rate is in nmol O min⁻¹ mg protein⁻¹. In some cases rates of H₂O₂ efflux and O₂ consumption could not be measured at all assay temperatures for mitochondria isolated from the same individual animal therefore values are expressed as mean ± SEM where the SEM was derived by standard error propagation procedures. Differences among these mean values were compared by 2-tailed Welch's t-test (p < 0.05 being considered significant).

Aerobic locomotion in both fish and mammals is largely fuelled by carbohydrate and lipid oxidation (Lauff and Wood, 1996, 1997; McClelland, 2004), as such, we evaluated the potential for mitochondrial ROS formation during the oxidation of either pyruvate or palmitoylcarnitine to reflect these respective fuels. In both cases saturating malate was also included to alleviate the trapping of intramitochondrial coenzyme-A as acetyl-CoA by the provisioning of oxaloacetate and to supply NADH for complex I via malate dehydrogenase. When compared at physiological temperatures, 15°, 25°, and 37°C for ectothermic fish, Bluefin tuna and rat respectively, there was a pattern of increasing capacity for ROS production as physiological temperature increased but the tuna mitochondrial ROS production at 25°C was comparable to the rat mitochondria at 37°C (Figure 1).

In all species the mitochondrial H₂O₂ production was sensitive to temperature (Figure 1) with similar sensitivity across species. At higher temperatures the rate of H₂O₂ production was increased. Overall the rates of H₂O₂ production by isolated Pacific bluefin tuna mitochondria were comparable to other fishes. When measured at a common assay temperature of 25°C, the physiological temperature for the bluefin tuna red muscle, rates of H₂O₂ production by tuna mitochondria were not different from mitochondria isolated from the other fishes (Figure 1). In all cases the rates at 25°C in fish mitochondria were markedly higher than the rate seen with rat muscle mitochondria with both carbohydrate and lipid based oxidative substrates (Figure 1). Within species we found no pattern of difference in rates of H₂O₂ formation between oxidation of pyruvate or palmitoylcarnitine (Figures 1A,B).

To evaluate if the differences seen in H₂O₂ production rates might be linked to mitochondrial respiratory capacity we measured the rates of respiration at physiological temperatures as well as at a common temperature of 25°C. Under conditions of phosphorylating respiration (presence of ADP) and non-phosphorylating respiration (absence of ADP phosphorylation) there was an overall tendency for increasing rates with increasing physiological temperature (Figure 2). At the common temperature of 25°C the rates of respiration with either pyruvate or palmitoylcarnitine (both supplemented with malate) were not different across fish mitochondria, with the exception of sturgeon being higher than trout with pyruvate and malate under phosphorylating conditions (Figure 2). Rates of respiration were generally higher for rat mitochondria. The respiratory control ratio (RCR) was not affected by assay temperature except for trout (Figure 2C) and at 25°C the RCR for Pacific bluefin tuna was not different from the other fishes. The stability of RCR values across temperatures suggests collapsing mitochondrial coupling efficiency with changing temperature is an unlikely explanation for differences in H₂O₂ production with assay temperature.

At a common assay temperature of 25°C the FEL for all fish species was markedly greater than for rat, with Pacific bluefin tuna being unexceptional in comparison to the other fish (Figure 3). For species where two test temperatures were used (all species but the tuna) there was a tendency for increasing FEL with higher assay temperature. This thermal sensitivity led to some ectothermic fishes having values for FEL that were not different from the rat at physiological temperatures (Figure 3), but the high levels of variation warrant some caution with interpretation of this result.

For all species examined the production of H₂O₂ by isolated muscle mitochondria was a function of assay temperature. Higher assay temperatures led to higher rates of production (Figure 1). However, across assay temperatures and using all substrate mixes tested, the rates of H₂O₂ production by isolated Pacific bluefin tuna mitochondria compared favorably with other fish mitochondria. Similarly, Pacific bluefin tuna mitochondria had similar respiration rates to most other fish red muscle mitochondria when measured at a common assay temperature irrespective of the physiological body temperature for the mitochondria (Figure 2). Our respiration rates with pyruvate also compare well with those for other teleosts over a range of both in vitro assay and physiological temperatures (Johnston et al., 1994, 1998) suggesting our mitochondrial preparations are reasonably consistent with those used in other studies on temperature effects on fish mitochondria.

A potential way to separate mitochondrial H₂O₂ production from the mitochondrial roles in oxygen consumption is to express the electron leakage to H₂O₂ as a fraction of the overall electron flux (FEL). Using the FEL we find no evidence for reduced potential for H₂O₂ formation relative to substrate oxidation rates; tuna mitochondria were similar to mitochondria from ectothermic fishes (Figure 3). The respiratory capacity, measured as ADP-stimulated respiration rate, was similar in Pacific bluefin tuna mitochondria to most other fish red muscle mitochondria. This similarity in respiration rate is consistent with no compensatory change in the endothermic tuna red muscle in the overall metabolic systems responsible for electron flux to oxygen either as respiration or leaking as mitochondrial H₂O₂ production. Of note, one could argue that the ADP-stimulated rate of oxygen consumption better reflects the overall amount of biochemical “machinery” in the mitochondrion, and thus the phosphorylating rate of respiration (state 3) is a better denominator to normalize H₂O₂ production; however, we find the same overall pattern is seen as that for FEL indicating that regardless of the respiration rate used to normalize H₂O₂ production all fish muscle mitochondria appear comparable at the same assay temperature (data not shown).

We included one representative mammal, the rat. Overall, mitochondria from all fish assayed appear to have greater potential for electron leak relative to their respiratory capacity (FEL) compared to the rat. The similarity of FEL across fishes in this study suggests fishes, and possibly other ectotherms, may have a propensity toward increased electron leakage compared to more derived endothermic species. This may represent an adaption to minimize the ROS induced damage that could occur at higher endogenous temperatures common in mammals and birds. This pattern warrants further investigation to determine if it holds more broadly across taxa. Contrasting endothermic and ectothermic representative birds and reptiles would be a particularly interesting comparison along these lines.

During aerobic locomotion both lipid and carbohydrate are important carbon sources for oxidation in teleost fishes (Lauff and Wood, 1996, 1997) and mammals (McClelland, 2004). This is consistent with relatively similar rates of pyruvate and fatty acid oxidation by isolated muscle mitochondria from teleosts as seen by us and others (Moyes et al., 1989, 1992; Chamberlin et al., 1991). Thus, pyruvate and palmitoylcarnitine were used as representative carbohydrate- and lipid-based oxidative substrates in the present study.

These physiologically important substrates were also chosen because they are known to produce H₂O₂ in isolated mitochondria (reviewed in Brand, 2010). Pyruvate and palmitoylcarnitine may contribute directly to mitochondrial ROS production via the matrix enzymes involved with their metabolism, as well as indirectly by producing NADH and QH₂ which reduce electron transport chain complexes I and III respectively. It has been reported that palmitoylcarnitine driven respiration in rodent mitochondria may have high capacity for H₂O₂ production compared to other NADH-generating substrates (St Pierre et al., 2002; Seifert et al., 2010); although more recent work found the rates comparable to that found with other NADH-generating substrates (Perevoshchikova et al., 2013). If lipid fuelled respiration was to lead to greater potential for H₂O₂ production then there is a possibility that the enhanced lipid oxidation proposed for high-performance fishes (Weber and Haman, 1996) may have consequences on mitochondrial H₂O₂ production. However, we broadly find comparable rates of H₂O₂ production, FEL, and ADP-stimulated respiration rates with isolated muscle mitochondria from several fishes and one mammal. As such, our evidence does not support a trade-off between selection for lipid or carbohydrate oxidation in muscle and potential for H₂O₂ production.

The elevated red muscle temperature in endothermic fishes should increase the potential for H₂O₂ formation but we see no evidence for compensatory decrease in H₂O₂ production in the Pacific Bluefin tuna, whereas we do see this in a representative endothermic mammal. Slow-twitch red muscle has a primary function in locomotion in these fish. This suggests that there is a trade-off in red, slow-twitch muscle between thermal effects that are selected for to improve muscle performance at elevated temperature (Altringham and Block, 1997), and the propensity toward mitochondrial H₂O₂ formation in the evolution of endothermy in fishes.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.